The Enhancer–Promoter-Mediated Wnt8a Transcription During Neurite Regrowth of Injured Cortical Neurons

Brain injuries can result from accidents, warfare, sports injuries, or brain diseases. Identifying regeneration-associated genes (RAGs) during epigenome remodeling upon brain injury could have a significant impact on reducing neuronal death and subsequent neurodegeneration for patients with brain injury. We previously identified several WNT genes as RAGs involved in the neurite regrowth of injured cortical neurons. Among them, the expression of the <i>Wnt8a</i> gene increased most significantly during neurite regrowth, indicating its potential to promote neuronal regeneration. In this study, we investigated the regulatory mechanism of <i>Wnt8a</i> transcription. An algorithm was developed to predict the novel enhancer regions of candidate genes. By combining active enhancer marks, histone H3 lysine 27 acetylation (H3K27ac), and histone H3 lysine 4 mono-methylation (H3K4me1), we identified a candidate enhancer region for <i>Wnt8a</i> located 1.7 Mb upstream and 0.1 Mb downstream of the <i>Wnt8a</i> gene. This region was organized into enhancers (Ens) 1-15. Enhancer RNA expression from the predicted En1-15 regions, DNA topological dynamics, and the activity of predicted enhancers were analyzed to validate the candidate active enhancers. Our findings showed that the En8, 9, 10, 14, and 15 regions expressed higher eRNAs during neurite regrowth. Notably, the En8-2 and En14-2 subregions showed significantly up-regulated H3K4me1 modification during neurite regrowth. Using chromatin conformation capture assays and enhancer-reporter assays, we delineated that the molecular regulation of <i>Wnt8a</i> transcription during neurite regrowth occurs through looped En8-promoter interplay.